Chronic lung diseases are a significant health and economic burden worldwide. For example, chronic obstructive lung disease (COPD) is the fourth leading cause of death in adults (Rennard S I (1998) “COPD: overview of definitions, epidemiology, and factors influencing its development,” Chest. 113:235S-241S), and lung cancer has the highest mortality of all cancers in both men and women (Alberg A J and Samet J M (2003) “Epidemiology of lung cancer,” Chest. 123:21S-49S). The common risk factor for both is cigarette smoking. However, only 10-15% of smokers develop COPD and/or lung cancer. Non-invasive efforts to identify biomarkers for such conditions have not been efficient or clinically effective.
Conventional methodologies typically provide for measuring biomarkers in lungs via invasive procedures such as bronchoscopy to obtain samples, carrying the associated costs, discomfort, and risks. Such biomarkers are needed for identifying the environmental factors in the generation and natural history of chronic lung diseases, and would allow for reliably following molecular events that are currently beyond detection using conventional methodologies.
Exhaled biomarkers could serve as a molecular and genetic signature, opening the doors for personalized medicine. Exhaled breath is an aerosol consisting mostly of water vapor, with smaller amounts of volatile, semi-volatile, and non-volatile molecules derived from the upper and lower portions of the respiratory system (Effros R M et al. (2005) “Epithelial lining fluid solute concentrations in chronic obstructive lung disease patients and normal subjects,” J. Appl. Physiol. 99:1286-1292; Horvath I et al. (2005) “Exhaled breath condensate: methodological recommendations and unresolved questions,” Eur. Respir. J. 26:523-548).
Cytokines are small, water-soluble signaling proteins produced by cells of the immune system to modulate responses of the immune system such as inflammation. Since inflammation is an underlying condition of many chronic diseases, exhaled cytokines may be considered biomarkers of pulmonary inflammation that could indicate the presence of lung diseases or provide information regarding the current status of the lungs. As such, non-invasive monitoring of lung inflammation through detection and measurement of cytokines in exhaled breath samples would be a promising new approach aimed at addressing the need for an improved understanding, treatment and management of chronic respiratory diseases such as lung cancer, asthma and COPD.
Thus, there has been great interest in the study of exhaled breath condensate (EBC), and in techniques for the collection and analysis of non-volatile compounds (e.g. cytokines) present in the respiratory lining fluid (RLF). Studies of exhaled breath suggest that humans generate fine particles during tidal breathing, but little is known of their origin in the respiratory system. Older studies of exhaled breath primarily detected particles larger than 1 μm due to less sensitive techniques, such as counting particles in photographs of coughs and sneezes (Jennison M W “Atomizing of Mouth And Nose Secretions into the Air as Revealed by High-Speed Photography, in Aerobiology Publication,” Washington, D.C.: American Association for the Advancement of Science, p. 106), culturing of indicator bacteria exhaled and impacted on plates (Duguid J (1945) “The numbers and the sites of origin of the droplets expelled during expiratory activities,” Edinburgh Med. J. 52:385-401), and counting slides or filters of exhaled dye droplets under a microscope (Id.; Loudon R G and Roberts R M (1967) “Droplet expulsion from the respiratory tract,” Am Rev Respir. Dis. 95:435-442). In such older studies, particles were typically detected only during coughs and sneezes, and not in breath exhaled during tidal breathing.
In more recent studies, it has been shown that approximately 98% of particles produced during tidal breathing are under 1 μm (Fairchild C I and Stampfer J F (1987) “Particle concentration in exhaled breath,” Am. Ind. Hyg. Assoc. J. 48:948-949; Papineni R S and Rosenthal F S (1997) “The size distribution of droplets in the exhaled breath of healthy human subjects,” J. Aerosol Med. 10:105-116; Edwards D A et al. (2004) “Inhaling to mitigate exhaled bioaerosols,” Proc. Natl. Acad. Sci. USA 101:17383-17388; Morawska L et al. (2008) “Size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities,” J. Aerosol. Sci. 40:256-269). For example, in a previous study of subjects infected with influenza, it was found that the subjects produced 67 to 8500 particles per liter of air, and that 87% of the particles were under 1 μm (Fabian P et al. (2008) “Influenza virus in human exhaled breath: an observational study,” PLoS ONE 3:e2691).
Such droplets can be generated by shear forces produced by air flow acting on the airway lining fluid and entraining particles composed of mucus, surfactant, and pathogens (King M et al. (1985) “Clearance of mucus by simulated cough,” J. Appl. Physiol. 58:1776-1782; Moriarty J A and Grotberg J B (1999) “Flow-induced instabilities of a mucus-serous bilayer,” J. Fluid Mech. 397:1-22), especially during cough (Leith D et al. (1986) “Cough” in M J Macklem (ed). Handbook of Physiology, The Respiratory System, Section 3, Vol. III, Part 1, Bethesda, Md.: American Physiological Society, pp. 315-336). It has been hypothesized that droplets are produced from the destabilization of the lining fluid during the reopening of collapsed small airways and alveoli during breathing (Edwards D A et al. (2004), supra., Proc. Natl. Acad. Sci. USA 101:17383-17388). Another study found that exhaled particle concentrations increased 4- to 18-fold when inhaling deeply and rapidly after a deep exhalation, hypothesizing that the opening of airways and alveoli blocked by fluid during inhalation is a significant source of particles (Johnson G R and Morawska L (2009) “The mechanism of breath aerosol formation,” J. Aerosol Med. Pulm. Drug Deliv. 22:229-237). Identifying the origin of these particles is important when interpreting studies of exhaled breath biomarkers, including cytokines (Shahid S K et al. (2002) “Increased interleukin-4 and decreased interferon-gamma in exhaled breath condensate of children with asthma,” Am. J. Respir. Crit. Care Med., 165:1290-1293; Garey K W et al. (2004) “Markers of inflammation in exhaled breath condensate of young healthy smokers,” Chest. 125: 22-26; Rosias P P et al. (2004) “Childhood asthma: exhaled markers of airway inflammation, asthma control score, and lung function tests,” Pediatr. Pulmonol. 38:107-114; Carpagnano G E et al. (2002) “Interleukin-6 is increased in breath condensate of patients with non-small cell lung cancer,” Int. J. Biol. Markers, 17:141-145; Leung T F et al. (2004) “Increased macrophage-derived chemokine in exhaled breath condensate and plasma from children with asthma,” Clin Exp Allergy, 34:786-791; and Rosias P et al. (2004) “Exhaled breath condensate: a space odessey, where no one has gone before,” Eur. Respir. J. 24:189-190), metals (Broding H C et al. (2009) “Comparison between exhaled breath condensate analysis as a marker for cobalt and tungsten exposure and biomonitoring in workers of a hard metal alloy processing plant,” Int. Arch. Occup. Environ. Health. 82:565-573; Goldoni M et al. (2008) “Chromium in exhaled breath condensate and pulmonary tissue of non-small cell lung cancer patients,” Int. Arch. Occup. Environ. Health, 81:487-493; Mutti A et al. (2006) “Exhaled metallic elements and serum pneumoproteins in asymptomatic smokers and patients with COPD or asthma,” Chest. 129:1288-1297), and pathogens such as viruses (Fabian P et al. (2008), supra., PLoS ONE 3:e2691; Huynh K N et al. (2008) “A new method for sampling and detection of exhaled respiratory virus aerosols,” Clin. Infect. Dis. 46:93-95) and bacteria (Fennelly K P et al. (2004) “Cough-generated aerosols of Mycobacterium tuberculosis: a new method to study infectiousness,” Am. J. Respir. Crit. Care Med. 169:604-609).
Collection of EBC samples non-invasively may be accomplished through means whereby a subject breathes normally into a chilled collection device that condenses and collects fluid samples. EBC samples consist of a mixture of three main components (Horvath I et al. (2005), supra., Eur. Respir. J. 26:523-548). The most abundant component (99%) of EBC samples is liquid water formed from the condensation of water vapor present in the warm exhaled air, saturated with water vapor as it leaves the respiratory tract. The second and third components of EBC samples are water-soluble volatile and non-volatile particles that are aerosolized from the respiratory lining fluid and are present in significantly smaller amounts than the water component of EBC samples (Horvath I et al. (2005), supra., Eur. Respir. J. 26:523-548; Kietzmann D et al. (1993) “Hydrogen peroxide in expired breath condensate of patients with acute respiratory failure and with ARDS,” Intensive Care Med. 19:78-81; Effros R M et al. (2002) “Dilution of respiratory solutes in exhaled condensates,” Am. J. Respir. Crit. Care Med. 165:663-669; Horvath I et al. (2009) “Exhaled biomarkers in lung cancer,” Eur. Respir. J. 34:261-275; Kazani S and Israel E (2010) “Exhaled breath condensates in asthma: diagnostic and therapeutic implications,” J. Breath Res. 4:047001; Loukides S et al. (2011) “Exhaled breath condensate in asthma: from bench to bedside,” Curr. Med. Chem. 18:1432-1443).
Unfortunately, the significant amount of liquid water present in EBC samples dilutes the inherently low concentrations of non-volatile biomarkers to levels that are at or below the detection threshold of methodologies utilizing conventional assays. Moreover, the inefficient collection of exhaled, nonvolatile submicron particles using conventional EBC collection methods, combined with assay sensitivity limitations currently being used, creates significant problems with reproducibility and validity of biomarker measurements (Horvath I et al. (2005), supra., Eur. Respir. J. 26:523-548; Kazani S and Israel E (2010) “Exhaled breath condensates in asthma: diagnostic and therapeutic implications,” J. Breath Res. 4:047001; Loukides S et al. (2011), supra., Curr. Med. Chem. 18:1432-1443; Sack U et al. (2006) “Multiplex analysis of cytokines in exhaled breath condensate,” Cytometry A. 69:169-172; Bayley D L et al. (2008) “Validation of assays for inflammatory mediators in exhaled breath condensate,” Eur. Respir. J. 31:943-948; Sapey E et al. (2008) “The validation of assays used to measure biomarkers in exhaled breath condensate,” Eur. Respir. J. 32:1408-1409). For example, the aerosol particle collection efficiency of conventional EBC devices is typically less than 25%.
As a result, conventional systems and methods for biomarker collection and analysis fail to provide sufficient sensitivity and efficiency for detecting cytokines and other non-volatile biomarkers in EBC. The availability of an effective, non-invasive system and methodology able to detect trace amounts of target biomarker(s) would open a new world of possibilities to the diagnosis and management of lung diseases and disorders.